FAU Institutional Repository http://purl.fcla.edu/fau/fauir This paper was submitted by the faculty of FAU’s Harbor Branch Oceanographic Institute. Notice: ©2000 Springer‐Verlag. This manuscript is an author version with the final publication available at http://www.springerlink.com and may be cited as: Pomponi, S. A., & Willoughby, R. (2000). Development of sponge cell cultures for biomedical application. In C. Mothersill & B. Austin (Eds.). Aquatic invertebrate cell culture. (pp. 323‐336). Berlin: Springer‐Verlag. 14 Development of sponge cell cultures for biomedical application Shirley A. Pomponi and Robin Willoughby 14.1 IMPORTANCE OF SPO GE CELL CULTURE The mari ne environment is a rich source of both biological and chemical diversity. During the past two deca des, there has been significant effort made in the discovery of novel, marine-derived, nat ural products with po tential for development as phar­ maceut icals, nut ritional supplements, cosmetics, agr ichemicals, molecular probes, enzymes and fine chemicals. Each of these classes of marine bioproducts has a potential multibillion-dollar mark et value (BioScience, 1996). Sponges (phylum Porifera) have been the most studied group for mari ne biopro­ ducts (Munro et al., 1999) and have yielded the greatest number of compo unds (I reiand et al., 1993). The emphasis on discovery of biolo gically active sponge metabolites is due to a number of factors. Sponges are among the most abunda nt and diverse groups of invertebrates present in benthic marine environment s world­ wide. There are ", 6,000 described species of sponges and perhaps twice as many undescribed species. Although sponge l arvae ~ 'pr~nktonic , adult sponges are sessile. It is not so diffieult to understand why sponges are a major source of cytoto xic, antibiotic, and anti-inflammatory compounds. During the course of their evolutionary history, they have evolved the abilit y to biosynthesize metabolites for defense against predati on , for inhibition of settlement or overgrowth by compet­ ing organisms, for contro l against infection by the microbial flora that filter through their bodies, for reproductive cues to conspecifics and for recognition of self and non- self (i.e. immune responses). Unfortunately, the roles that most biologically active metabolites play in the marine organisms that synthesize them are largely unknown. An und erstanding of the roles of these metabolites in nature could, in fact, lead to a more rational approach to the discovery of commercially useful marine bioproducts. 324 Development of sponge cell cultures for biomedical applications [Ch.14 As a result of the potential commercial importance of sponge-derived natural products, there has been a renewed effort to understand their phylogenetic relation­ ships, chemical ecology, and physiology in an attempt to predict the occurrence or determine trends in the production of novel secondary metabolites, as well as to understand the mechanisms which stimulate the production of these compounds. In addition, with the awareness that natural populations of sponges (and other marine organisms) cannot support the harvests predicted for supply of sufficient quantities of bioactive compounds for development of the bioproducts, research is in progress to develop alternative supply methods, such as chemical synthesis, aquaculture, cell culture and recombinant production. The objective of research in progress in our laboratory is to establish cell lines of bioactive marine invertebrates that can be used as models to study in vitro produc­ tion of bioactive metabolites and the factors which control expression ofproduction. We hypothesize that understanding the molecular mechanisms involved in growth regulation and bioactive metabolite production in these organisms will lead to the development of genetically engineered cell-lines capable of overexpression of bioac­ tive natural products. Perhaps more important, however, is the development of cell lines of these 'simple' metazoans to study basic cell physiology and molecular biology that may be applied to understanding more complex metazoan systems, including humans.' 14.2 SPONGE CELL CULTURES 14.2.1 Cell culture versus tissue culture Traditionally, cell cultures are differentiated from tissue or organ cultures on the basis of a few key criteria: their lineage, their potential for cell division, and their uniformity (Freshney, 1987). Tissue or organ cultures are derived from explants or fragments of the tissue or organ; they retain the architecture characteristic of that tissue or organ, and they are comprised-of differentiated cells with limited capacity for cell proliferation. Cell cultures may be derived from explants or dissociated cell suspensions; they have the capacity for proliferation, and cells with similar rates of growth will predominate to form lines of homogeneous cell types. Typically, normal (mammalian) cells will form a monolayer and must remain attached to the substrate to proliferate, while only haemopoietic, malignant or transformed cells will grow in suspension (Freshney, 1987). With an increase in the understanding of basic metabolic processes at the cellular level in mammalian cell cultures has come a transition to focusing on understanding these processes in differentiated, three­ dimensional populations of cells. A similar trend is occurring in sponge cell culture. While there is still much to learn about basic cellular and molecular processes in sponge cells, there is much to gain from research using both undifferentiated and differentiated cells in both cell cultures and 'tissue cultures' (e.g. primmorphs, Custodio et al., 1998; Muller et al., 1999; Muller and Custodio, this volume). We have the opportunity to compare differential expression in differentiated versus Sec. 14.3] Establishment of ceU cultures 325 undifferentiated cells, in homogeneous versus heterogeneous/integrated populations and in cell-culture monolayers versus three-dimensional cultures which preserve the architecture of the 'functional' adult sponge . Because of their cellular level of organization, sponges can be easily dissociated into single cells (Wilson, 1907), which will reaggregate to form a functional sponge (i.e. an aggregate with differentiated cells). This basic ability makes sponges the ideal organism to use as an in vitro model. A cell culture is most likely an equilibrium of multipotent stem cells, undifferentiated but committed precursor cells, and mature differentiated cells. The equilibrium will shift according to changes in culture con­ ditions (Freshney, 1987). The challenge is to understand both the position of the cell in its lineage (i.e. whether it is an uncommitted stem cell, a committed but undiffer­ entiated precursor or a mature differentiated cell) and to understand the conditions that will induce the cells to differentiate or dedifferentiate. For sponge cells, many of these questions remain to be addressed. The development of a cell line may simply appear to involve the application of existing concepts and technology developed for the culture of other heterotrophic eukaryotic cells, such as vertebrate cells (Ham and McKeehan, 1979). The discovery of growth factors, cytokines, and hormones, together with the development of basal nutrient media for specific cell types (Hayashi and Sato , 1976) has made it virtually routine to culture a wide variety of vertebrate cell types. But even in the case of vertebrate cells, culture of specialized cell types beyond primary culture (e.g. epi­ thelial cells) was only achieved by empirical means during the last 30 years (Kaighn and Lechner, 1984). In contrast, very little is known about culture requirements for marine sponges or, for that matter, for marine invertebrates in general. Development of marine invertebrate cell lines (i.e. continuously replicating normal or transformed cells) has been problematic. The status of the science and the problems encountered are reviewed by Rinkevich (1999). 14.3 ESTABLISHMENT OF CELL CULTURES 14.3.1 Selection of appropriate species for in vitro research .-;- . ~ ~ " .. ~ - I- - If the objective of sponge cell culture is the mvitro production of bioactive metabo­ lites or the evaluation 'of cellular and molecular events involved in the production of these compounds, selection of the appropriate sponge species is essential. A number of secondary metabolites reported from marine invertebrates have been proposed to have a microbial origin. This is generally based upon: (1) the presence of the compounds as trace metabolites in the source organism; and /or (2) close structural similarity to compounds reported either from microbial sources or from widely divergent taxa. However, in only a few cases have the compounds been unequivo­ cally localized in microbial associates (e.g. Unson et al., 1994; Bewley et al., 1996). There are equally compelling data to indicate that bioactive metabolites are localized in sponge cells (Faulkner et al., 1993; Garson et al., 1994, 1998; Uriz et al., 1996a, b; Flowers et al., 1998). 326 Development of sponge cell cultures for biomedical applications leh.14 The prim ary species selected for our cell-culture development research is Teichax­ inella morchella (= Axinella corrugata) (Demospongiae: Axinellida). The antitumour compound stevensine (Albitzi and Faulkner, 1985) constitutes approximately 0.5 per cent of the wet weight of this sponge (Pomponi et al.. 1997a). Similar compounds have been found in related axinellid taxa but do not occur randomly in unrelated taxa. A